Hypocitraturia is an important cause of kidney stones. Urinary citrate excretion is regulated principally by the reabsorption of citrate in the proximal tubule. Proximal tubular citrate reabsorption is regulated at two steps: i) apical membrane citrate uptake; and 2) mitochondrial citrate uptake and metabolism. The proposed studies will examine four major aims regarding citrate transport by the proximal tubule. In Aim 1, we will clone the renal apical membrane Na/citrate cotransporter by expression cloning using an oocyte expression system. We will then characterize the transporter with regard to structure, function, and tissue distribution, in order to definitively determine that the clone corresponds to the apical membrane transporter. We will also clone other isoforms expressed in kidney cortex or intestine. Lastly, we will clone the human apical membrane Na/citrate transporter cDNA and gene, in order to allow genetic studies and definition of promoter function. In Aim 2, we will define the molecular basis for chronic regulation of the apical membrane Na/citrate cotransporter. Chronic acidosis and chronic K deficiency have both been demonstrated to cause chronic adaptations in the transporter that persist when the transporter is assayed in vitro. The present studies will examine the chronic regulation of apical membrane Na/citrate transporter: a) activity; b) protein abundance by western blot; c) protein distribution by cell fractionation and western blot; d) mRNA abundance by northern blot; and e) transcriptional rate by nuclear runoff. in Aim 3, we will examine whether there is chronic regulation of mitochondrial citrate transport and/or metabolism. Using the same chronic models of regulation, we will examine whether there are changes in mitochondrial function that persist in vitro. Mitochondrial citrate uptake will be measured as well as the activities of a number of key members of the tricarboxylic acid cycle. Once again, we will use western blotting to determine whether the abundance of any of the TCA cycle enzymes has been altered, northern blotting to determine whether the abundance of their respective mRNAs has been altered, and nuclear runoff studies to examine transcriptional rate. Lastly, using quantitative morphometry, we will examine whether these chronic conditions are associated with alterations in inner mitochondrial membrane surface area. In Aim 4, we will examine whether overactivity of the apical membrane Na/citrate transporter is responsible for familial hypocitraturia. In preliminary studies performed during the previous grant period, we have identified a number of families with frequent kidney stones, in which the proband has idiopathic hypocitraturia. We will examine other members of these families, as well as investigating other families. We will then explore whether any mutations are present in the apical membrane Na/citrate transporter gene that cosegregate with idiopathic hypocitraturia.